4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone
(Synonyms: 4-甲基亚硝胺基-1-3-吡啶基-1-丁酮(NNK),NNK) 目录号 : GC46607
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone是一种主要存在于烟草制品中的强效烟草特异性致癌物，对α7烟碱型乙酰胆碱受体（α7 nAChR）具有很高的亲和力，在小细胞肺癌（SCLCs）中的EC50值为0.03μM，在肺神经内分泌细胞（PNECs）中的EC50值为0.005μM。
Cas No.:64091-91-4
Sample solution is provided at 25 µL, 10mM.
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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone is a potent tobacco-specific carcinogen primarily found in tobacco products. It has a high affinity for α7 nicotinic acetylcholine receptors (α7 nAChR), with EC50 values of 0.03μM in small cell lung carcinoma (SCLCs) and 0.005μM in pulmonary neuroendocrine cells (PNECs)[1][2]. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone can induce the occurrence of tumors in various tissues, such as the lungs and liver. Studying its carcinogenic mechanisms helps to develop strategies for the prevention and treatment of tobacco-related cancers[3][4][5][6]. In tobacco harm reduction research, the content of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and the levels of its metabolites are often used as important indicators for assessing safety[7].
In vitro, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone interacts with the α7 nAChR and activates the Raf-1/MAPK signaling cascade, promoting cancer progression and cancer cell proliferation. In cultured PNECs, a 6-day exposure to 100pM 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone led to an 11.9-fold increase in Raf-1 protein and a 2.8-fold increase in MAPK protein. A single dose of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (100pM) caused time-dependent increases in Raf-1 and MAPK proteins, peaking at 4.8-fold for Raf-1 at 5min and 2.9-fold for MAPK at 15min. PNEC showed significant increases in DNA synthesis in response to 100pM 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, peaking at 150min[2]. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (100pM) incubation for 6 days induced proliferation of NCI-H69 lung cancer cells may occur through phosphorylation of Bcl2 and c-Myc to facilitate their cooperation[3]. In mono-culture system, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (50, 100, and 200μg/mL) treatment for 24h decreased human bronchial epithelial cells (Beas-2B) proliferation, increased apoptosis, caused G1 phase arrest, and increased DNA damage in a dose-dependent manner. However, these cytotoxic effects were significantly reduced in the co-culture model of Beas-2B and macrophages cells (U937) in transwell, likely due to changes in cytokine expression and activation of related pathways[4].
In vivo, C3H and A/J mice were treated with three doses of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (100mg/kg/d) via intraperitoneal injection on three alternate days. C3H mice did not develop noticeable lung tumors within 7 months after 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment. In contrast, A/J mice developed visible lung tumors with an adenomatous pattern. Their immune responses were significantly suppressed, including AFC response, and anti-CD3/CD28 antibody-induced intracellular calcium concentration changes. Moreover, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment led to a sustained increase in the expression of α7-nAChR and COX-2 in the lungs of A/J mice[5]. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone was administered to A/J mice in the drinking water during 7 weeks at doses of 9.2 or 3.1mg/mouse. Mice that received a total dose of 9.2mg 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone per mouse had an average of 15.7 ± 2.7 lung tumors, whereas those that received 3.1mg 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone per mouse had only 1.2 ± 0.3 tumors per mouse[6]. Male ICR mice were exposed to 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (0.5mg/mouse) and sodium arsenite (0, 10, or 20mg/kg) daily via gavaging for 10 days and their urine was collected at day 10 for 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolite analysis. The results suggested that sodium arsenite increased 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism by up-regulation of CYP2A expression and activity leading to an increased 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism and DNA adducts[7].
References:
[1] Lisa A Peterson L A, Stephen B Stanfill S B, Stephen S Hecht S S. An update on the formation in tobacco, toxicity and carcinogenicity of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Carcinogenesis. 2024 May 19;45(5):275-287.
[2] Schuller H M, Plummer H K, Jull B A. Receptor-mediated effects of nicotine and its nitrosated derivative NNK on pulmonary neuroendocrine cells. Anat Rec A Discov Mol Cell Evol Biol. 2003 Jan;270(1):51-8.
[3] Jin Z H, Gao F Q, Flagg T, Deng X M. Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation. J Biol Chem. 2004 Sep 17;279(38):40209-19.
[4] Zhou J X, Zou H X, Liu Y Q, et al. Acute cytotoxicity test of PM2.5, NNK and BPDE in human normal bronchial epithelial cells: A comparison of a co-culture model containing macrophages and a mono-culture model. Toxicol In Vitro. 2022 Dec:85:105480.
[5] Boroujerdi R S, Sopori M L. Early manifestations of NNK-induced lung cancer: role of lung immunity in tumor susceptibility. Am J Respir Cell Mol Biol. 2007 Jan;36(1):13-9.
[6] Castonguay A, Pepin P, Stoner G D.Lung tumorigenicity of NNK given orally to A/J mice: its application to chemopreventive efficacy studies. Exp Lung Res. 1991 Mar-Apr;17(2):485-99.
[7] Lee H L, Chang L W, Wu J P, et al. Enhancements of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone) metabolism and carcinogenic risk via 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone/arsenic interaction. Toxicol Appl Pharmacol. 2008 Feb 15;227(1):108-14.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone是一种主要存在于烟草制品中的强效烟草特异性致癌物，对α7烟碱型乙酰胆碱受体（α7 nAChR）具有很高的亲和力，在小细胞肺癌（SCLCs）中的EC50值为0.03μM，在肺神经内分泌细胞（PNECs）中的EC50值为0.005μM[1][2]。4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone可以诱导多种组织（如肺和肝）中肿瘤的发生。研究其致癌机制有助于开发预防和治疗烟草相关癌症的策略[3][4][5][6]。在烟草减害研究中，4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone的含量及其代谢物的水平常被用作评估安全性的关键指标[7]。
在体外实验中，4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone与α7 nAChR相互作用并激活Raf-1/MAPK信号级联反应，从而促进癌症进展和癌细胞增殖。在培养的PNECs中，暴露于4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone（100pM） 6天后，Raf-1蛋白增加了11.9倍，MAPK蛋白增加了2.8倍。单剂量的4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone（100pM）导致Raf-1和MAPK蛋白随时间增加，Raf-1在5min时达到4.8倍的峰值，MAPK在15min时达到2.9倍的峰值。PNECs对100pM 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone的反应表现为DNA合成显著增加，在150分钟时达到峰值[2]。4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone（100pM）孵育6天可诱导NCI-H69肺癌细胞增殖，这可能是通过Bcl2和c-Myc的磷酸化来促进它们的协同作用[3]。在单培养体系中，4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone（50、100和200μg/mL）处理24小时可降低人支气管上皮细胞（Beas-2B）的增殖，增加凋亡，导致G1期阻滞，并以剂量依赖性方式增加DNA损伤。然而，在transwell中共培养Beas-2B和巨噬细胞（U937）的模型中，这些细胞毒性效应显著降低，这可能是由于细胞因子表达的变化和相关途径的激活[4]。
在体内实验中，C3H和A/J小鼠每隔三天腹膜内注射三次剂量4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone（100mg/kg/d）。在4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone处理后7个月内，C3H小鼠未出现明显的肺肿瘤。相比之下，A/J小鼠出现了腺瘤样模式的可见肺肿瘤。它们的免疫反应显著受到抑制，包括AFC反应和抗CD3/CD28抗体诱导的细胞内钙浓度变化。此外，4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone处理导致A/J小鼠肺部α7-nAChR和COX-2的表达持续增加[5]。在7周内，通过饮用水给予A/J小鼠4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone，剂量为每只小鼠9.2或3.1mg。接受9.2mg 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone的小鼠平均每只有15.7±2.7个肺肿瘤，而接受3.1mg 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone的小鼠平均每只有1.2±0.3个肿瘤[6]。雄性ICR小鼠每天通过灌胃暴露于4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone（0.5mg/只小鼠）和亚砷酸钠（0、10或20mg/kg），连续10天，并在第10天收集尿液进行4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone代谢物分析。结果表明，亚砷酸钠通过上调CYP2A的基因表达和活性增加了4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone的代谢，导致4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone代谢和DNA加合物增加[7]。
| Cell experiment [1]: | |
Cell lines | human bronchial epithelial cells (Beas-2B) |
Preparation Method | First, macrophages cells (U937) were treated with PMA (Phorbol-12-myristate 13-acetate) for 24h to induce differentiation into macrophage-like cells. Beas-2B cells were cultured in the lower room of the (1.2 × 105 cells/well) transwell system. Beas-2B cells grown in mono-culture or co-culture systems with U937 were observed respectively under the microscopy after 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone exposure for 24h to better understand its effects on cell growth, adhesion and morphology. |
Reaction Conditions | 50, 100 and 200μg/mL; 24h |
Applications | In mono-culture system, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone decreased human bronchial epithelial cells (Beas-2B) proliferation, increased apoptosis, caused G1 phase arrest, and increased DNA damage in a dose-dependent manner. However, these cytotoxic effects were significantly reduced in the co-culture model of Beas-2B and macrophages cells (U937) in transwell, likely due to changes in cytokine expression and activation of related pathways. |
| Animal experiment [2]: | |
Animal models | C3H and A/J mice |
Preparation Method | Mice were injected intraperitoneally with three doses of 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (100mg/kg/d in 0.1ml PBS) on three alternate days. Control animals received an equivalent volume of PBS. |
Dosage form | 100mg/kg/d; i.p.; 3 times |
Applications | C3H mice did not develop noticeable lung tumors within 7 months after 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment. In contrast, A/J mice developed visible lung tumors with an adenomatous pattern. Their immune responses were significantly suppressed, including AFC response, and anti-CD3/CD28 antibody-induced intracellular calcium concentration changes. Moreover, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment led to a sustained increase in the expression of α7-nAChR and COX-2 in the lungs of A/J mice. |
References: | |
| Cas No. | 64091-91-4 | SDF | |
| 别名 | 4-甲基亚硝胺基-1-3-吡啶基-1-丁酮(NNK),NNK | ||
| 化学名 | 4-(methylnitrosoamino)-1-(3-pyridinyl)-1-butanone | ||
| Canonical SMILES | O=C(CCCN(C)N=O)C1=CC=CN=C1 | ||
| 分子式 | C10H13N3O2 | 分子量 | 207.2 |
| 溶解度 | DMF: 30 mg/ml, DMSO: 25 mg/ml, Ethanol: 25 mg/ml, | 储存条件 | Store at -20°C |
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1 mg | 5 mg | 10 mg |
| 1 mM | 4.8263 mL | 24.1313 mL | 48.2625 mL |
| 5 mM | 0.9653 mL | 4.8263 mL | 9.6525 mL |
| 10 mM | 0.4826 mL | 2.4131 mL | 4.8263 mL |
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